L-Forms, Genes, and Division Timing

1. L-Forms, Genes, and Division Timing Cell Shape Journal Club Tristan Ursell February 2011

3. First, a bit of Terminology CWDB – cell wall deficient bacteria - often a mode of immune system evasion L-Form – four different types

4. Forming L-Forms Happens in liquid and on solid media FtsZ independent cell division ‘observed’ Work usually concentrates on Gram-positives Different profiles of gene expression Mutations generally acquired through lengthy selection Can sometimes revert back to cell-wall form, but conditions are poorly understood Are they Lamarckian?

5. Forming L-Forms Brain-Heart Infusion Media 1% agar, 10% sucrose, 0.125% MgSO4 6mg(10000units)/ml Penicillin G Grown 37C, aerobically, 48-72 hours Look for ‘fried egg’ colony morphology About 1 in 10^4 – 10^5 convert Able to propagate in Pen G Eventually revert in LB (to varying degrees)

6. L-Forms, it’s what’s for Breakfast

7. So what did they do? Grew up L-form colonies in BHI+Sucrose+PenG Grew control colonies in BHI+Sucrose Microarrayed each to measure differential gene expression – found 450 genes expressed in a greater than 4-fold ratio Vast majority (427/450) up-regulated. Surprise! Of the 450, %58 of unknown function

8. What is a Microarray?

9. What is a Microarray? Cy3 or Cy5 DNA dye

10. What is a Microarray? Run mRNA in triplicate. Spot cDNA at least twice. Watch for expressed sequence tag (EST) overload Uses pre-defined cDNA set hence why sequencing is important! Requires strict definitions of statistical significance mRNA is not whole story! They used a 4-fold change criteria arbitrary and not sound with a small sample size Statistical analysis is a big problem List of identified genes depends on analysis

11. Key Up-regulated Genes DNA repair Osmoregulation LPS biosynthesis Heat shock Phage Shock Envelope Stress Drug efflux pumps too many others to list

12. The Clever Part Use the non-essential deletion subset of the Keio Collection (3985) Found 52 mutants that could not form L-Forms Some simply didn’t grow (24/52) Others formed few colonies (low conversion) Others formed small colonies (low growth) Now let’s examine what happened in these cases.

14. The Valiant Never Taste of Death But Once Phosphorelays DNA repair/SOS response Colonic acid biosynthesis exopolysaccharide Lipo-proteins smpA implicated in virulence of Salmonella and OM integrity yfgl forms part of the membrane insertion pathway Drug efflux/ABC transporters largest superfamily of proteins! iron scavenging (siderophores) 10^-24M/L in vivo (enterobactin made by E. coli 10^52/M) Iron homeostasis PBP1B (transglycosylase)

15. Cowards Die Many Times Before Their Death Groups 2 and 3 Some co-grouped with Group 1 Those that did not: DNA replication LPS synthesis Energy metabolism Siderophore hydrolysis Claim: these genes help with L-form growth, not conversion to L-form.

16. Complementation Using 4 Group 1 Mutants for: rcsB (colanic acid) Fur (iron homeostasis) ruvA(DNA repair) smpA(OM lipoprotein) Upon high copy number complementation - no L-form recovery Upon low copy number complementation - all mutants regained L-form ability

20. Their Tools Typical K12 strain – using both phase and cytoplasmic GFP microscopy. Custom made sub-pixel resolution contour algorithm (which I did not look at in detail). Fitting of the contour to the (assumed) cell shape. Test different models of the rate and shape of division region with improved resolution.

21. Four Basic Assumptions The cell is a cylinder with hemi-spherical caps. At intermediate times during division, the constriction consists of two symmetric, incomplete hemispheres. The cell width is constant throughout the cell cycle. The area of the constriction grows at a constant rate. - What does that imply?

22. Four Basic Assumptions 4) The area of the constriction grows at a constant rate. - What does that imply?

23. Model of Growth R = cell radius r = constriction radius t = time τc = time of constriction start τg = time between successive divisions Comes from a simple geometric derivation of the shape and the constant division area growth.

24. Model of Growth R = cell radius r = constriction radius t = time τc = time of constriction start τg = time between successive divisions Comes from a simple geometric derivation of the shape and the constant division area growth. Now fit to sub-pixel resolution shape data!

25. Some Simple Observations The observable time of division initiationlags behind the fit value by ~30%.

26. Some Simple Observations The observable time of division initiationlags behind the fit value by ~30%.

27. Length Dynamics

28. Length Dynamics Claim: Growth speeds up after division initiation because both the cylinder and the new pole are now growing.

29. More Subtle Claim: Constriction time is a constant, division time is variable. Or cells with shorter doubling times start constriction earlier.

30. More Subtle Constriction time is inversely correlated to birth length – indicating a length compensation mechanism.

31. What does this need? Better statistics – it would be nice to see distributions of these parameters. Do the same thing – now including fusion proteins from the divisome. Would be cool to see which parts of division antibiotics are affecting.